CD63 in Cancer: CD63 is a Type III membrane protein of the tetraspanin family whose 30 current members are characterized by the presence of four transmembrane segments. Several groups independently identified CD63, using antibodies raised to whole cell preparations of activated platelets, granulocytes, and melanoma cells. Cloning of the respective cDNAs of their cognate glycoprotein antigens led to the recognition that the different antigens were one and the same molecule. The Sixth International Workshop on Leukocyte Typing (1996) subsequently categorized these antibodies as CD63 antibodies. Prior to the 1996 Workshop, CD63 was known by multiple names (melanoma 1 antigen, ocular melanoma-associated antigen, melanoma associated antigen ME491, lysosome-associated membrane glycoprotein 3, granulophysin, melanoma-associated antigen MLA1), which were sometimes related to the antibodies that led to its partial characterization and identification. Thus, CD63 was also designated as antigen ME491 (MAb ME491), neuroglandular antigen (MAbs LS59, LS62, LS76, LS113, LS140 and LS152), Pltgp40 (MAbs H5C6, H4F8 and H5D2), human bone marrow stromal cell antigen (MAb 12F12), osteoprogenitor-specific marker (MAb HOP-26), and integrin-associated protein (MAb 6H1). Other antibodies that were found to cross react with human CD63 were 8-1H, 8-2A (cross-reactivity with ME491), NKI/C-3 and NKI/black-13 (Vennegoor et al. Int J Cancer 35(3):287-95 (1985); Vennegoor and Rumke, Cancer Immunol Immunother. 23(2):93-100 (1986); Demetrick et al., J Natl Cancer Inst 84(6):422-9 (1992); Wang et al., Arch Ophthalmol. 110(3):399-404 (1992)). Work with the rabbit polyclonal antibody RaC3, raised against the immunoaffinity-purified NKI/C3 antigen, revealed the target protein as a core polypeptide with an apparent molecular weight around 20 kDa and heavily post-translationally modified by N-linked carbohydrate (Gruters et al., Cancer Res 49(2):459-65 (1989)).
CD63 was initially cloned from a melanoma cDNA library using MAb ME491, one of a number of antibodies raised against a preparation the SK Mel 23 human cutaneous melanoma cell line and screened for binding to melanoma cells. Immunoprecipitation from 125I-lactoperoxidase-labelled melanoma cells revealed a 30-60 kDa protein present at the cell surface. The antigen recognized by this antibody was shown to be a protein highly post-translationally modified. By immunohistochemistry the antibody was found to recognize melanoma cells in tumor tissue but not the surrounding normal looking cells, thus suggesting that it recognized a potentially tumor-specific antigen determinant (Atkinson et al., Hybridoma 4, 243-255 (1984)). This antibody also stained melanoma cells in 87% of uveal melanoma cases and balloon cells in 86% of the cases where the latter were present. In this study staining of the normal occular tissue was variable, and positive in only a few of the normal cases and only rarely in the morphologically normal melanocytes (Folberg et al., Arch Ophthalmol 103(2):275-9 (1985)). In a separate experiment monoclonal antibodies MAb6-F1, MAb8-1H and MAb8-2A, raised also against the SK Mel 23 cell line, were shown to recognize the same antigen and to display an immunohistochemical staining pattern very similar to that obtained with MAb ME491. In addition these antibodies stained liver metastatic tumor tissue, in patients with primary choroidal melanoma but did not stain normal hepatocytes. In another study of human melanoma biopsies it was shown that the reactivity of MAb ME491 appeared to be inversely correlated with melanoma progression. The reactivity of the ME491 antibody was low in normal melanocytes, higher in the early stages of melanoma progression (dysplastic nevi and radial growth phase (RGP) tumors) and decreased or even absent in more advanced melanoma tumors such as those in the vertical growth phase (VGP) and in metastatic tumors. Another monoclonal antibody (MAb 4A3) raised against primary human uveal melanoma cells was found to specifically recognize an antigen present in these cells, and revealed only background levels of binding to lymphocytes from healthy individuals. The antigen(s) detected by this antibody, by Western immunoblotting of melanoma tissue, consist(s) of a doublet with an approximate apparent molecular weight of 55 kDa, suggesting that the antigen recognized by this antibody was not the same as that recognized by the antibodies that were clustered as anti-CD63 (Damato et al., Invest Ophthalmol Vis Sci 27(9):1362-7 (1986)).
CD63 was also found and partially characterized in human platelets using monoclonal antibodies raised against thrombin-activated platelets (MAbs 2.28, 2.19, 5.15 and 5d10). These antibodies detected an activation-dependent platelet membrane 53 kDa glycoprotein, as demonstrated by the increased number of binding sites (more than 10 fold) upon thrombin activation. In competition assays these antibodies blocked each other binding, suggesting that they recognized the same or spatially close antigen determinants. Results from platelet aggregation experiments revealed that these antibodies per se did not cause platelet aggregation, nor did they interfere with the aggregation induced by adenosine di-phosphate (ADP), thrombin, collagen, ristocetin and epinephrin. Electron microscopy data suggested that in resting platelets these antibodies recognized an antigen localized in lysosome membranes. Immunohistochemistry data indicated that these antibodies recognized an antigen present in restricted regions of spleen, lymph nodes, thymus and in endothelial cells. In another study the MAb 2.28 also labelled internal granules in resting platelets and in megakaryocytes and endothelial cells, and in the latter two it co-localized with antibodies to the enzyme cathepsin D, a known marker of lysosomal compartments. Follow up studies with antibody clustering and expression cloning, led to the identification of the antigen recognized by this antibody as CD63, and further confirmed its presence in lysosomal compartments, where it co-localized with the compartment-specific markers LAMP-1 and LAMP-2. Cloning of this molecule identified it as CD63 and allowed its inclusion in the tetraspanin family.
Expression of CD63 was detected in many different tissues and cell types. At the cellular level it was found to be associated with the plasma membrane and also with intracellular late endosomal vesicular structures. Cell activation led, in certain cases, to increased surface expression by mobilization of intracellular stores of CD63. CD63 was also found to co-localize, and physically associate, with MHC class II in B-lymphocytes, particularly in endosomes, in exosomes involved in exporting MHC class II complexes to the surface, and in secreted vesicles. CD63 was found to interact with other members of the tetraspanin family, such as CD9, CD81, CD111 (integrin chain αM,L,X), CD18 (integrin chain β2), CD49c (VLA-3 or integrin chain α3), CD49d (integrin chain α4), CD49f (VLA-6 or integrin chain α6) and CD29 (integrin chain β1), in a variety of cell types including B- and T-lymphocytes, neutrophils, breast cancer and melanoma cells.
The role of CD63 in cancer has been unclear. Although CD63 was initially discovered by several independent groups to be involved in diverse events such as platelet and granulocyte activation, MHC class II-dependent antigen presentation, integrin-dependent cell adhesion and motility, and tumor progression in certain types of cancers, its function has yet to be fully elucidated. Even though current evidence supports its role in a variety of cellular physiological events, it is not clear if these functions are independent of each other or if there is an underlying common cellular mechanism in which CD63 is involved.
Several groups have investigated the association between CD63 and the progression of certain types of tumors, particularly melanomas. A number of other anti-CD63 monoclonal antibodies, in addition to Mab ME491, were developed for immunohistochemical (IHC) staining of cancer samples obtained from patients with tumors at various stages of progression. It was observed that decreased staining, interpreted by the authors as most likely reflecting decreased expression of CD63, correlated with advanced progression and with metastatic characteristics of the tumors. A more recent study, also described a significant correlation between the apparent decreased expression levels (after quantitation of mRNA) of several members of the tetraspanin protein family, including CD63, and the in vitro invasiveness of several mammary carcinoma-derived cell lines. Another study identified CD63, by differential display, in cultured breast cancer cells subjected to estrogen deprivation. This indicated that CD63 expression can be steroid-hormone regulated and that altered CD63 abundance and/or function might also be associated with breast tumor progression.
By contrast, work with anti-CD63 monoclonal antibody MAb FC-5.01 revealed that its reactive epitope was variably expressed in different normal tissues. Although this antibody was found to recognize CD63, it did not distinguish between early and more advanced stage melanomas, including metastatic melanomas (unlike MAb ME491), which suggested that the CD63 antigen was present in these more advanced tumors, but that some of its epitopes may have been masked in the cells from tumors at different stages. This might have been due to altered post-translational modifications of the core CD63 polypeptide, or to the interaction of CD63 with other molecules, which might have affected the availability of specific epitopes for antibody recognition and binding. These results supported the observation, described by Si and Hersey, Int J Cancer 54(1):37-43 (1993), that staining with the anti-CD63 MAb NKI-C3, did not distinguish between tissue sections from melanomas at different stages of progression, such as primary, radial growth phase, vertical growth phase, and metastatic melanomas. Although in other studies (Adachi et al., J Clin Oncol 16(4): 1397-406 (1998); Huang et al., Am J Pathol 153(3):973-83 (1998)) analysis of mRNA from breast, and from non-small-cell lung cancers, by quantitative PCR, revealed that for two tetraspanin family members (CD9 and CD82) there was a significant correlation between their expression levels and tumor progression and patient prognosis, no such correlation was found for CD63, in that its expression was similar in all the samples. As a result of these, apparently conflicting, results, there is lack of strong and consistent data that would definitively demonstrate the association of CD63 with cancer.
To date very few in vivo studies have attempted to establish a link between CD63 and an eventual tumor suppressor function of this molecule. In one of these studies, human CD63-overexpressing H-ras-transformed NIH-3T3 cells, injected both subcutaneously and intraperitoneally into athymic mice, revealed a decreased malignant/tumorigenic phenotype, as indicated by decreased tumor size and metastatic potential as well as by increased survival time, when compared to the behavior of the parental non-CD63-overexpressing cells. This suggested that the presence of human CD63 in the transformed cells might suppress their malignant behavior. More recently, work with a transgenic mouse model expressing human CD63, and developed to induce tolerance to CD63, indicated that tumor growth of an injected human CD63-MHC class I (H-2Kb) co-transfected murine melanoma cell line could be inhibited, and survival increased, upon immunization with human CD63 fused to vaccinia virus. It was suggested by the authors that the therapeutic effect was T-lymphocyte-dependent, and that endogenous anti-CD63 antibodies did not appear to be involved in this protective effect, since tumor growth inhibition only occurred when animals were injected with the CD63-MHC class I co-transfected cells and not with the CD63-only transfected cell line. This interpretation was supported by the fact that in wild type animals, pre-immunized with purified human CD63 and shown to have developed anti-human CD63 antibodies, there was no protective effect against tumor cell growth. Work described by Radford et al., Int J Cancer 62(5):631-5 (1995) using the KM3 cell line, initially thought to be of human origin but later characterized as being of rat lineage, transfected with human CD63, suggested that expression of this protein decreased the growth and metastastic potential of these cells, relative to that observed using the parental non-transfected KM3 cells, when injected intradermally into athymic mice, although there was no significant difference between the in vitro growth rates of the various transfected and non-transfected cell lines. These observations distinguished the potential effect of CD63 from that of other tumor suppressor genes known to affect both the in vivo and the in vitro growth rates of tumor cells. Furthermore, addition of the anti-CD63 monoclonal antibody ME491, which was found to have a functional effect on the same cells by decreasing their random motility in an in vitro assay (Radford et al., J Immunol 158(7):3353-8 (1997)), did not impact their in vitro growth rates.
This study also described the observation that CD63 may promote migration in response to extracellular matrix (ECM)-derived chemoattractants, such as laminin, fibronectin, collagen and vitronectin, and that this effect may be mediated by the functional involvement of β1-type integrins, although antibodies to the integrins were unable to block these effects. However, there appeared to be an antagonistic effect between the role of vitronectin-mediated signaling (a known ligand for the integrin αvβ5) and that of the signaling mediated by other ECM components such as fibronectin, laminin and collagen on CD63 transfected cells. This suggested that under specific conditions, in the presence of ECM components, expression of CD63 may lead to decreased migration, and that this may be dependent on a fine balance between adhesion and motility. In another study, an anti-CD63 monoclonal antibody (MAb 710F) enhanced the adhesion and spreading of PMA-treated HL-60 cells, while another anti-CD63 monoclonal antibody (MAb 2.28), promoted a similar effect, but only on a much smaller fraction of the cell population, and only when added in much larger amounts. These results showed that although many antibodies to CD63 have been developed, their functional effects can be quite different.
Tetraspanins may also be involved in cell proliferation. Oren et al. Mol Cell Biol 10(8):4007-15 (1990) described anti-proliferative effects of the murine MAb 5A6, that recognizes CD81 (TAPA-1), on lymphoma cell lines. In another study, ligation of CD37 in human T-lymphocytes with antibodies blocked CD37-induced proliferation. More recently, a study with an animal model deficient in the expression of CD37 (CD37 knockout) revealed that T lymphocytes from this animal were hyperproliferative compared to those from wild type animals in response to concanavalin A activation and CD3/T cell receptor engagement. It was therefore proposed that a functional role in cell growth and proliferation might be a common feature of the tetraspanin family. Recent studies with hepatoblastoma and hepatocellular carcinoma cells revealed that engagement of these cells with anti-CD81 monoclonal antibodies led to activation of the Erk/MAP kinase pathway. This signaling pathway has been shown to be involved with cell growth and proliferation events. In parallel work, transfected cell lines overexpressing human CD81 displayed increased proliferation relative to the mock-transfected control cells. Therefore, available evidence has pointed to a role of the tetraspanins in general, and of CD63 in particular, in events associated with cell growth proliferation and with cell adhesion/motility. These two types of cellular events are currently the target of intense research as both play a central role in tumor progression and metastasis.
Amino acid sequence determination and analysis did not reveal homology between tetraspanins and other protein families, or with any previously characterized functional modules, nor has it suggested any previously known enzymatic activity. As a result it has been very difficult to investigate the role of this family of proteins in the modulation of signal transduction pathways. However, the evidence generated using tetraspanin-specific reagents that led to changes in cellular physiology, and which were intimately dependent on the modulation of signal transduction pathways, suggests that tetraspanins have signal transduction properties. CD63 was shown to associate, both physically and functionally, with a number of molecules that are themselves either enzymes involved in the generation of secondary messenger signals, or are associated physically and/or functionally with such enzymes.
Experiments designed to dissect the mechanism controlling the interaction of human neutrophils with endothelial cells, which is one of the initial steps of the inflammatory response, revealed that pre-treatment of neutrophils with several anti-CD63 monoclonal antibodies (AHN-16, AHN-16.1, AHN-16.2, AHN-16.3 and AHN-16-5) promoted their adhesion to cultured endothelial cell layers. Furthermore this effect was strongly dependent on the presence of calcium ion (Ca2+), a well-known modulator of many intracellular signaling pathways and which was restricted to a specific period of time during which the cells were exposed to the stimulating antibodies. After longer exposure to the antibody, adhesion of the neutrophils to the endothelial cells became insensitive to the later addition of Ca2+, therefore implicating a dynamic and temporally regulated (transitory) event. In addition, CD63 was found to physically interact with the CD11/CD18 protein complex, and reagents that specifically targeted this complex mediated a modulatory signal. In this study CD63 was also found to be physically associated with, or to be part of, a complex that included the enzyme tyrosine kinases Lck and Hck. These enzymes are members of a class of proteins that play a central role in mediating intracellular regulatory signals upon activation of specific surface receptors and are part of cascades of signaling pathways that result in cell-specific physiological changes. Another study suggested that co-ligation of tetraspanins (including CD63) with monoclonal antibodies could enhance the phosphorylation or activity of the enzyme focal adhesion kinase (FAK) that was induced by adhesion of MDA-MB-231 breast cancer cells to collagen substrate. This pointed to a direct involvement of CD63 (and of other tetraspanin family members) in the modulation of integrin-mediated tyrosine kinase signaling pathways. Other signaling pathways that may functionally intersect with the presence and ligation of surface CD63 by the anti-CD63 monoclonal antibody MAb 710F appear to be those dependent on modulation of phosphorylation by the enzyme protein kinase C (PKC), another well known modulator of intracellular signaling pathways. In this context, enhancement of adhesion and of morphological changes in the myeloid cell line HL-60 by MAb 710F was dependent on pre-treatment of the cells with phorbol myristate acetate (PMA) although the temporal involvement of PKC was not conclusively demonstrated. However, later work by an independent group demonstrated that PMA-induced HL-60 differentiation was PKC-activity dependent since the molecule Ro31-8220, a specific inhibitor of this enzyme, blocked the effect of PMA.
Further evidence supporting the association of CD63, and other tetraspanin family members, with signal transduction pathways, arose from work that described a physical association, either direct or as part of a supramolecular complex, between CD63 (and also CD53) molecules with tyrosine phosphatase activity. In this study, immunoprecipitate complexes isolated with anti-CD63 antibodies were shown to be associated with tyrosine phosphatase activity, although unlike for CD53, which was shown to associate with the tyrosine phosphatase CD45, it was not possible to identify the CD63-associated phosphatase. More recently several members of the tetraspanin family were also found to be associated with a type II phosphatidylinositol 4-kinase (type II PI 4-K) (Berditchevski et al., J Biol Chem 272(5):2595-8 (1997)). This interaction appeared to be very specific since it was only identified for CD9, CD63, CD81, CD151 and A15/TALLA, and it was not observed to occur with CD37, CD52, CD82, or NAG-2. In addition, the association between tetraspanin family members and PI-4K was mutually exclusive since each PI-4 kinase-containing complex was limited to a single tetraspanin family member. CD63-PI-4 kinase complexes, in particular, were found, almost entirely, in intracellular compartments in lipid raft-like domains, unlike those formed with the other tetraspanin members. This observation suggested that this CD63 fraction, found to interact with the PI-4 kinase, might have been involved in specific intracellular events (Claas et al., J Biol Chem 276(11):7974-84 (2001)) related to, or dependent from, phosphoinositide biosynthesis pathways, which are well known for their involvement in the regulation of membrane trafficking (endocytosis and exocytosis) and of cytoskeleton reorganization, in addition to their function as secondary messenger molecules (Martin Annu. Rev. Cell. Dev Biol 14:231-64 (1998)).
The direct and important involvement of all the enzymes, that CD63 was found until now to be directly associated with, in the regulation of signaling pathways provided further evidence in support of the association of CD63 with the modulation of signal transduction pathways, either as a regulator or as an effector molecule downstream from the activity of these enzymes.
Elucidation of the mechanisms that lead to tumor progression is a very difficult and complex endeavor frequently marked by apparently contradictory observations and, as a result, it is rare that those observations successfully translate into effective therapies. In view of what is currently known about the association of CD63 with tumor progression and metastasis and with signal transduction mechanisms, it is possible that its function may be altered, in tumor cells.
Development of antigen-specific reagents with cytotoxic effects on tumor cells, that bind cells expressing the recognized antigen(s) and which by themselves, or associated with other molecules, have cellular and in vivo physiological activity such that these reagents inhibit tumor cell growth, progression and metastasis, without significant deleterious effects on normal cell populations, would be extremely beneficial as a potential therapeutic and or diagnostic tool.
Recently, new data has pointed to an important mode of action of CD63 in the regulation of normal cell physiology, and that when altered may have important impact on the behavior of the cells under pathological conditions, including in cancer.
MAb antibody Fc-5.01, known to cause internalization of CD63 in breast cancer cells was used to determine the levels of surface expression and internalization of CD63 in human dendritic cells (DCs) (Mantezazza et al., Blood 104(4):1183-90 (2004)). CD63 was found to localize both at the cell surface and intracellularly, in co-localization with endosomal and lysosomal markers. The intact antibody, and its Fab fragments were able to induce internalization of CD63. Simultaneously internalization of CD63 promoted by Fc-5.01 resulted in decreased surface expression of several integrin molecules, CD11b, CD18, CD29 and α5, but not of β3 or of HLA-II molecules. Results from a chemotaxis assay revealed that this antibody, and others that recognize other members of the tetraspanin family of proteins, caused an increase in the number of cells that migrated across a membrane barrier towards chemoattractants. In these cells (immature DCs) yeast phagocytosis, which is mediated by β1,3-glycan receptors was accompanied by a decrease in the levels of cell surface CD63 but not of the tetraspanins CD9, CD81 and CD82, nor of HLA-II molecules. On the other hand, internalization induced by Dextran-FITC, which is mediated by the macrophage mannose receptor (MMR) did not result in decreased CD63 surface expression or of CD9, CD81, CD82, HLA-I and HLA-II molecules. Therefore it would appear that CD63 is associated with specific receptors, sometimes physically as in the case of the β1,3-glycan receptor dectin-1, and participate in the internalization events. The fact that the surface expression of several integrin molecules is decreased upon antibody-induced internalization of CD63 also suggests that such a CD63-dependent event can have a significant impact on the cell surface receptor composition and thus impact the physiology of such cell populations as demonstrated by the effect on the DC migration assay.
In another study, the internalization of membrane type-1 metalloproteinase (MT1-MMP) was found to be affected by CD63. In this study FLAG-tagged MT1-MMP internalized and acquired a diffuse cytoplasmic distribution that was accompanied by a decrease in its cell surface levels. Addition of chlorquine, a known lysosomal proteiase inhibitor, partially inhibited this internalization-dependent disappearance of cell surface MT1-MMP, and simultaneously altered the internalization-dependent cytoplasmic distribution in such a way that MT1-MMP remained associated with CD63 positive internal granule-type structures. Co-transfection of cells with MT1-MMP and CD63 resulted in decreased cell surface levels of this metalloprotease, which was not dependent on the overall levels of MMP activity, since an inhibitor of these molecules, BB94, did not have any impact on this decrease, while chlorquine did. This observation suggested that increased CD63 expression may accelerate the turnover/internalization/degradation of MT1-MMP. The increased internalization/degradation of MT1-MMP depended on the direct interaction between MT1-MMP and CD63. This type of function was further supported by previous observations that CD63 directly interacted with the μ2 and μ3 subunbits of the adaptor proteins AP-2 and AP-3 respectively and which are involved in protein sorting to endosomes and lysosomes. It was also previously shown that the cytoplasmic tail of MT1-MMP was important for the internalization of this molecule and that this event played an important role in the regulation of its invasion-promoting activity. MT1-MMP is also considered to play important roles in the invasion of malignant tumor cells. Therefore it is possible that regulation of its overall levels may depend on its interaction and internalization by associating with CD63.
In another recent publication (Xu et al., Embo J 23(4):811-22 (2004)) a genetic screen for Drosophila eye-enriched genes that might be involved in retinal degeneration identified a large number of genes among which that of a tetraspanin-like molecule named ‘sunglasses’ (‘sun’). The closest-related mammalian protein was the tetraspanin CD63. And similarly to CD63, ‘sun’ was found to be enriched in lysosomes, as suggested by immunoelectron microscopy. Results from this work suggested that ‘sun’ participates in the normal downregulation of Rh1 signaling, independent of the arrestin mediated mechanism that is typical of other G-protein coupled receptors. In addition ‘sun’ was important not only in the regular turnover of activated Rh1 but also, and possibly dependent on this event, had a significant impact in the maintenance of rhabdomers' structure, which resulted in a dramatic sun-dependent retinal degeneration in the mutant flies. Together the data implicates this homologue of mammalian CD63 in the normal trafficking-dependent turnover of proteins and abnormalities in its expression/function result in physiological abnormalities.
Another publication on the role of CD63 in receptor internalization described the co-localization of the β-subunit of the gastric ion pump H,K-ATPase with this tetraspanin in COS cells co-transfected with both molecules. In this study it was found also that the H,K-ATPase β-subunit underwent a CD63 expression-dependent enhancement of internalization and of localization to lysosome-like cytoplasmic granular structures.
All the data from the work described above suggested that CD63 was also involved in the normal turnover of cell-surface molecules, by participating in their internalization and lysosomal-dependent degradation, thus participating in the control of the normal cell physiology. It is possible, therefore, that manipulation of this function of CD63 might be an important tool to control events dependent on the activity of specific molecules, or groups of molecules, whose surface expression or function is either altered or contributes to abnormal cell behavior in pathological conditions such as cancer.
Until now, no anti-CD63 antibodies, or other reagents that specifically targeted CD63-expressing cells, were reported and shown to have a simultaneous impact on the in vitro and on the in vivo growth characteristics of tumor cells, and also on the survival time of animal models of tumor cell growth.
Monoclonal Antibodies as Cancer Therapy: Each individual who presents with cancer is unique and has a cancer that is as different from other cancers as that person's identity. Despite this, current therapy treats all patients with the same type of cancer, at the same stage, in the same way. At least 30 percent of these patients will fail the first line therapy, thus leading to further rounds of treatment and the increased probability of treatment failure, metastases, and ultimately, death. A superior approach to treatment would be the customization of therapy for the particular individual. The only current therapy which lends itself to customization is surgery. Chemotherapy and radiation treatment cannot be tailored to the patient, and surgery by itself, in most cases is inadequate for producing cures.
With the advent of monoclonal antibodies, the possibility of developing methods for customized therapy became more realistic since each antibody can be directed to a single epitope. Furthermore, it is possible to produce a combination of antibodies that are directed to the constellation of epitopes that uniquely define a particular individual's tumor.
Having recognized that a significant difference between cancerous and normal cells is that cancerous cells contain antigens that are specific to transformed cells, the scientific community has long held that monoclonal antibodies can be designed to specifically target transformed cells by binding specifically to these cancer antigens; thus giving rise to the belief that monoclonal antibodies can serve as “Magic Bullets” to eliminate cancer cells. However, it is now widely recognized that no single monoclonal antibody can serve in all instances of cancer, and that monoclonal antibodies can be deployed, as a class, as targeted cancer treatments. Monoclonal antibodies isolated in accordance with the teachings of the instantly disclosed invention have been shown to modify the cancerous disease process in a manner which is beneficial to the patient, for example by reducing the tumor burden, and will variously be referred to herein as cancerous disease modifying antibodies (CDMAB) or “anti-cancer” antibodies.
At the present time, the cancer patient usually has few options of treatment. The regimented approach to cancer therapy has produced improvements in global survival and morbidity rates. However, to the particular individual, these improved statistics do not necessarily correlate with an improvement in their personal situation.
Thus, if a methodology was put forth which enabled the practitioner to treat each tumor independently of other patients in the same cohort, this would permit the unique approach of tailoring therapy to just that one person. Such a course of therapy would, ideally, increase the rate of cures, and produce better outcomes, thereby satisfying a long-felt need.
Historically, the use of polyclonal antibodies has been used with limited success in the treatment of human cancers. Lymphomas and leukemias have been treated with human plasma, but there were few prolonged remission or responses. Furthermore, there was a lack of reproducibility and there was no additional benefit compared to chemotherapy. Solid tumors such as breast cancers, melanomas and renal cell carcinomas have also been treated with human blood, chimpanzee serum, human plasma and horse serum with correspondingly unpredictable and ineffective results.
There have been many clinical trials of monoclonal antibodies for solid tumors. In the 1980s there were at least four clinical trials for human breast cancer which produced only one responder from at least 47 patients using antibodies against specific antigens or based on tissue selectivity. It was not until 1998 that there was a successful clinical trial using a humanized anti-Her2/neu antibody (Herceptin®) in combination with cisplatin. In this trial 37 patients were assessed for responses of which about a quarter had a partial response rate and an additional quarter had minor or stable disease progression. The median time to progression among the responders was 8.4 months with median response duration of 5.3 months.
Herceptin® was approved in 1998 for first line use in combination with Taxol®. Clinical study results showed an increase in the median time to disease progression for those who received antibody therapy plus Taxol® (6.9 months) in comparison to the group that received Taxol® alone (3.0 months). There was also a slight increase in median survival; 22 versus 18 months for the Herceptin® plus Taxol® treatment arm versus the Taxol® treatment alone arm. In addition, there was an increase in the number of both complete (8 versus 2 percent) and partial responders (34 versus 15 percent) in the antibody plus Taxol® combination group in comparison to Taxol® alone. However, treatment with Herceptin® and Taxol® led to a higher incidence of cardiotoxicity in comparison to Taxol® treatment alone (13 versus 1 percent respectively). Also, Herceptin® therapy was only effective for patients who over express (as determined through immunohistochemistry (IHC) analysis) the human epidermal growth factor receptor 2 (Her2/neu), a receptor, which currently has no known function or biologically important ligand; approximately 25 percent of patients who have metastatic breast cancer. Therefore, there is still a large unmet need for patients with breast cancer. Even those who can benefit from Herceptin® treatment would still require chemotherapy and consequently would still have to deal with, at least to some degree, the side effects of this kind of treatment.
The clinical trials investigating colorectal cancer involve antibodies against both glycoprotein and glycolipid targets. Antibodies such as 17-1A, which has some specificity for adenocarcinomas, has undergone Phase 2 clinical trials in over 60 patients with only 1 patient having a partial response. In other trials, use of 17-1A produced only 1 complete response and 2 minor responses among 52 patients in protocols using additional cyclophosphamide. To date, Phase III clinical trials of 17-1A have not demonstrated improved efficacy as adjuvant therapy for stage III colon cancer. The use of a humanized murine monoclonal antibody initially approved for imaging also did not produce tumor regression.
Only recently have there been any positive results from colorectal cancer clinical studies with the use of monoclonal antibodies. In 2004, ERBITUX® was approved for the second line treatment of patients with EGFR-expressing metastatic colorectal cancer who are refractory to irinotecan-based chemotherapy. Results from both a two-arm Phase II clinical study and a single arm study showed that ERBITUX® in combination with irinotecan had a response rate of 23 and 15 percent respectively with a median time to disease progression of 4.1 and 6.5 months respectively. Results from the same two-arm Phase II clinical study and another single arm study showed that treatment with ERBITUX® alone resulted in an 11 and 9 percent response rate respectively with a median time to disease progression of 1.5 and 4.2 months respectively.
Consequently in both Switzerland and the United States, ERBITUX® treatment in combination with irinotecan, and in the United States, ERBITUX® treatment alone, has been approved as a second line treatment of colon cancer patients who have failed first line irinotecan therapy. Therefore, like Herceptin®, treatment in Switzerland is only approved as a combination of monoclonal antibody and chemotherapy. In addition, treatment in both Switzerland and the US is only approved for patients as a second line therapy. Also, in 2004, AVASTIN® was approved for use in combination with intravenous 5-fluorouracil-based chemotherapy as a first line treatment of metastatic colorectal cancer. Phase III clinical study results demonstrated a prolongation in the median survival of patients treated with AVASTIN® plus 5-fluorouracil compared to patients treated with 5-fluourouracil alone (20 months versus 16 months respectively). However, again like Herceptin® and ERBITUX®, treatment is only approved as a combination of monoclonal antibody and chemotherapy.
There also continues to be poor results for lung, brain, ovarian, pancreatic, prostate, and stomach cancer. The most promising recent results for non-small cell lung cancer came from a Phase II clinical trial where treatment involved a monoclonal antibody (SGN-15; dox-BR96, anti-Sialyl-LeX) conjugated to the cell-killing drug doxorubicin in combination with the chemotherapeutic agent TAXOTERE®. TAXOTERE® is the only FDA approved chemotherapy for the second line treatment of lung cancer. Initial data indicate an improved overall survival compared to TAXOTERE® alone. Out of the 62 patients who were recruited for the study, two-thirds received SGN-15 in combination with TAXOTERE® while the remaining one-third received TAXOTERE® alone. For the patients receiving SGN-15 in combination with TAXOTERE®, median overall survival was 7.3 months in comparison to 5.9 months for patients receiving TAXOTERE® alone. Overall survival at 1 year and 18 months was 29 and 18 percent respectively for patients receiving SNG-15 plus TAXOTERE® compared to 24 and 8 percent respectively for patients receiving TAXOTERE® alone. Further clinical trials are planned.
Preclinically, there has been some limited success in the use of monoclonal antibodies for melanoma. Very few of these antibodies have reached clinical trials and to date none have been approved or demonstrated favorable results in Phase III clinical trials.
The discovery of new drugs to treat disease is hindered by the lack of identification of relevant targets among the products of 30,000 known genes that unambiguously contribute to disease pathogenesis. In oncology research, potential drug targets are often selected simply due to the fact that they are over-expressed in tumor cells. Targets thus identified are then screened for interaction with a multitude of compounds. In the case of potential antibody therapies, these candidate compounds are usually derived from traditional methods of monoclonal antibody generation according to the fundamental principles laid down by Kohler and Milstein (1975, Nature, 256, 495-497, Kohler and Milstein). Spleen cells are collected from mice immunized with antigen (e.g. whole cells, cell fractions, purified antigen) and fused with immortalized hybridoma partners. The resulting hybridomas are screened and selected for secretion of antibodies which bind most avidly to the target. Many therapeutic and diagnostic antibodies directed against cancer cells, including Herceptin® and RITUXIMAB, have been produced using these methods and selected on the basis of their affinity. The flaws in this strategy are twofold. Firstly, the choice of appropriate targets for therapeutic or diagnostic antibody binding is limited by the paucity of knowledge surrounding tissue specific carcinogenic processes and the resulting simplistic methods, such as selection by overexpression, by which these targets are identified. Secondly, the assumption that the drug molecule that binds to the receptor with the greatest affinity usually has the highest probability for initiating or inhibiting a signal may not always be the case.
Despite some progress with the treatment of breast and colon cancer, the identification and development of efficacious antibody therapies, either as single agents or co-treatments, has been inadequate for all types of cancer.
Prior Patents:
U.S. Pat. No. 5,296,348 teaches methods for selecting monoclonal antibodies specific for cancer cell surface antigens that are internalizing, and for identifying monoclonal antibodies having anti-transcriptional and/or anti-replicational effects on cell metabolism. By way of example the ME491 antibody was shown to internalize in W9, WM35, WM983 melanoma cells, and SW948 colorectal carcinoma cells. In addition ME491 antibody was shown to decrease transcription and cell proliferation in SW948 cells. The patent application US20030211498A1 (and its related applications: WO0175177A3, WO0175177A2, AU0153140A5) allege a method of inhibiting the growth or metastasis of an ovarian tumor with an antibody that binds an ovarian tumor marker polypeptide encoded by an ovarian tumor marker gene selected from among a group that includes CD63 antigen. Serial analysis of gene expression using ovarian cancer was carried out to identify ovarian tumor marker genes which lead to the identification of CD63 as a candidate. The patent application WO02055551A1 (and its related application CN1364803A) alleges a new polypeptide-human CD63 antigen 56.87. The patent application CN1326962A alleges a new polypeptide-human CD63 antigen 14.63. The patent application CN1326951A alleges a new polypeptide-human CD63 antigen 15.07. The patent application CN1351054A alleges a new polypeptide-human CD63 antigen 11.11. These patents and patent applications identify CD63 antigens and antibodies but fail to disclose the isolated monoclonal antibody of the instant invention, or the utility of the isolated monoclonal antibody of the instant invention.
The gene encoding the ME491 polypeptide antigen was cloned and the sequence was received for publication on Feb. 24, 1988 (Can Res 48:2955, 1988, Jun. 1); the gene encoding CD63 was cloned and the sequence published in February 1991 (JBC 266(5):3239-3245, 1991) and the publication clearly indicated the identity of ME491 with CD63.
WO2004041170.89 (Sequence ID No.: 89, priority filing date: 29 Jun. 2004), WO2003068268-A2 (Sequence ID No.: 1, priority filing date: 13 Feb. 2003 (2003WO-EP001461); other priority date: 14 Feb. 2002 (2002 GB-00003480)), WO2003057160-A29 (Sequence ID No.: 40, priority filing date: 30 Dec. 2002 (2002WO-US041798); other priority date: 2 Jan. 2002 (2002US-0345444P)) all allege polypeptides that have 100 percent sequence homology to CD63.
WO2003016475-A2 (Sequence ID No.: 9787& 12101, priority filing date: 14 Aug. 2002 (2002WO-US025765); other priority date: 14 Aug. 2001 (2001 US-0312147P) allege polypeptides that have 100 percent sequence homology with 237 amino acids of 238 amino acids comprising CD63.
WO2003070902-A2 (Sequence ID No.:27, priority filing date: 18 Feb. 2003 (2003WO-US004902); other priority date: 20 Feb. 2002 (2002US-0358279P)) allege polypeptides that have 94 percent sequence homology with 224 amino acids of 238 amino acids comprising CD63.
EP1033401-A2 (Sequence ID No.: 4168& 4913, priority filing date: 21 Feb. 2000 (2000EP-00200610); other priority date: 26 Feb. 1999 (99US-0122487P)) allege polypeptides that have 100 percent sequence homology with 205 amino acids and with 94 amino acids of 238 amino acids comprising CD63, respectively.
WO200257303-A2 (Human prey protein for Shigella ospG#26, priority filing date: 11 Jan. 2002 (2002WO-EP000777); other priority date: 12 Jan. 2001 (2001US-0261130P)) allege polypeptides that have 100 percent sequence homology with 130 amino acids of 238 amino acids comprising CD63.
WO200055180-A2 (Sequence ID No.: 756, priority filing date: 08 Mar. 2000 (2000WO-US005918); other priority date: 12 Mar. 1999 (99US-0124270P)) allege polypeptides that have 99 percent sequence homology with 127 amino acids of 238 amino acids comprising CD63.
WO200200677-A1 (Sequence ID No.:3203, priority filing date: 07 Jun. 2001 (2001WO-US018569); other priority date: 7 Jun. 2000 (2000US-0209467P)) allege polypeptides that have 97 percent sequence homology with 132 amino acids of 238 amino acids comprising CD63.
WO9966027-A1 (Large extracellular loop sequence from human CD63 protein, priority filing date: 15 Jun. 1999 (99WO-US013480); other priority date: 15 Jun. 1998 (98US-0089226P)) allege polypeptides that have 100 percent sequence homology with 99 amino acids of 238 amino acids comprising CD63.
WO200270539-A2 (Sequence ID No.: 1207, priority filing date: 5 Mar. 2002 (2002WO-US005095); other priority date: 5 Mar. 2001 (2001 US-00799451)) allege polypeptides that have 86 percent sequence homology with 102 amino acids of 238 amino acids comprising CD63.
EP1033401-A2 (Sequence ID No.: 4169, 21 Feb. 2000 (2000EP-00200610); other priority date: 26 Feb. 1999 (99US-0122487P)) allege polypeptides that have 100 percent sequence homology with 74 amino acids of 238 amino acids comprising CD63.
These patent applications identify polypeptides that have varying sequence homology to CD63 antigen. In most cases these application also allege antibodies and antibody derivatives to the corresponding polypepide and their homologs but fail to disclose the isolated monoclonal antibody of the instant invention, or the utility of the isolated monoclonal antibody of the instant invention for the treatment of human lung, prostate and colon cancer or other human cancers. Importantly, all the above applications were filed after the publication of the sequence of the polynucleotide encoding CD63.